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Microbiology IISulfate reduction
Christopher Bräsenaus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
Lecture Plan
17.10. 2017 Mikrobielle Physiologie I ‐ Energetik Bräsen
24.10. 2017 Mikrobielle Physiologie II – Einige Prinzipien und Mechanismen im zentralen Kohlenstoffmetabolismus
Bräsen
31.10. 2017 Keine Vorlesung Bräsen
07.11. 2017 Mikrobielle Physiologie III – Nitrat‐Atmung Bräsen
14.11. 2017 Mikrobielle Physiologie IV – Acetogenese und der Acetyl‐CoA/Kohlenmonoxid Dehydrogenase‐Weg
Bräsen
21.11. 2017 Mikrobielle Physiologie V – Anaerobe Nahrungskette und Methanogenese Bräsen
28.11. 2017 Mikrobielle Physiologie VI – Sulfate Reduktion Bräsen
05.12. 2017 Antibiotika (Penicillium notatum) Meckenstock
12.12. 2017 Mikroorganismen in der Umwelt (Geobacter metallireducens) Meckenstock
19.12. 2017 Mikrobielles Wachstum (Elusimicrobium minutum) Meckenstock
09.01. 2018 Mikrobielle Fortbewegung (Thioploca) Meckenstock
16.01. 2018 Viren (T4) Meckenstock
23.01. 2018 Geschichte der Mikrobiologie Meckenstock
30.01. 2018 Wrap up/Ausweichtermin Meckenstock/Bräsen
Redox potentials
ΔG0‘ = ‐ n F ΔE0‘
Brock Mikrobiologie © Pearson 2013
Anaerobic food chain
Brock Mikrobiologie © Pearson 2013
Methane is a major end product of anaerobic biomass degradation only in anoxic environments where the concentrations of sulphate, nitrate, Mn(IV) or Fe(III) are low. In the presence of these electron acceptors, methanogenesis is out‐competed by anaerobic respiration, mainly for thermodynamic reasons. (Thauer RK et al. (2008) Nature Reviews Microbiology 6, 579‐591)
Sulfate reduction
Muyzer G & Stams AJM, 2008
• Competition of sulfate reducers with syntrophic methanogenic communities andacetogens for fermentation products (lactate, propionate, acetate, H2 in anaerobicenvironments)
• Sulfate reducers with broader substrate spectrum than e.g. methanogens
• Higher affinity and lower threshold for H2
→ sulfate reducers outcompete methanogens at elevated SO42‐ concentrations e.g. in
marine sediments
Redox potentials
ΔG0‘ = ‐ n F ΔE0‘
Brock Mikrobiologie © Pearson 2013
Sulfate reducers
• In five bacterial lineages, twoarchaeal
• anaerobic• Ubiquitous in habitats where
SO42‐ is present
• Seawater ~28 mM SO42‐
Muyzer G & Stams AJM, 2008
Sulfate reducers
Incomplete oxidizers
Sulphate reducing reaction ΔG0‘ group
SO42‐ + 4 H2 + H+ → HS‐ + 4 H2O ‐151.9 autotrophs Many sulfate reducers,
e.g. Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)
SO42‐ + Acetate → HS‐ + 2 HCO3
‐
(also: fatty acids, hydrocarbons, aromatic compounds→ Acetyl‐CoA)‐47.6 Complete oxidizers
(heterotrophs)Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)Many others
0.75 SO42‐ + propionate → 0.75 HS‐ + 2 acetate + HCO3
‐ +0.25 H+
0.5 SO42‐ + butyrate→ 0.5 HS‐ + 2 acetate + 0.5 H+
0.5 SO42‐ + lactate→ 0.5 HS‐ + acetate + HCO3
‐
‐37.7
‐27.8
‐80.2
Incompleteoxidizers(heterotrophs)
Desulfovibrio
Three main physiological groups:
Sulfate reduction(chemolitoautotrophic)
4 H2 SO42‐
4 H2O HS‐
net ~ 2 ATP8 [H]
Oxidative part Reductive part
Via Electron transportphosphorylation (ETP)
CO2 fixation via reductive acetyl‐CoA/CO dehydrogenase pathway, some via reductive citric acid cycle
Problems:Sulfate transportSulfate activation Energy consuming processes
4 H2 + SO42‐ + H+ HS‐ + 4H2O ΔG0‘ = ‐152 kJ/mol SO4
2‐ red.
Redox potentials
ΔG0‘ = ‐ n F ΔE0‘
Redox potential of SO42‐/HSO3
‐ toonega ve → cannot be reduced byNAD(P)H (or ferredoxinred)
SO42‐has to be activated at the
expense of ATPAdenylation of SO4
2‐ to APS
Brock Mikrobiologie © Pearson 2013
Sulphate activation
Organic sulfur (Cystein, Methionine, etc.) – E. coliSulphate esters
Dissimilatory sulphatereduction – excretionOrganic sulfur (Cystein, Methionine, etc.) – B. subtilisSulphate esters
Anhydride bond
SO42‐ activation energy consuming
Brock Mikrobiologie © Pearson 2013, modified
Sulfate reduction(chemolitoautotrophic)
4 H2 APS
4 H2O HS‐~ > 2 ATP
8 [H]
Oxidative part Reductive part
Via Electron transportphosphorylation (ETP)
CO2 fixation via reductive acetyl‐CoA/CO dehydrogenase pathway, some via reductive citric acid cycle
Problems:Sulfate transportSulfate activation Energy consuming processes
SO42‐
ATPPPi
AMP
Corresponds to 2 ATP equivalents whichhave to be invested
HSO3‐
ΔG0‘ = ‐152 kJ/mol SO42‐ red.
Sulfate reduction(chemolitoautotrophic)
4 H2 APS
4 H2O HS‐~ > 2 ATP (ETP)
8 [H]
Oxidative part Reductive part
CO2 fixation via reductive acetyl‐CoA/CO dehydrogenase pathway, some via reductive citric acid cycle
Problems:Sulfate transportSulfate activation Energy consuming processes
SO42‐
ATPPPi
AMP
HSO3‐
ATP‐Sulfurylase
APS reductase
Sulfite reductase
ΔµH+
Sulfate reduction(chemolitoautotrophic)
APS reductase Sulfite reductaseATP‐Sulfurylase
Soluble, cytoplasmic enzymes
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
Periplasmic hydrogenase
Sulfate reduction(chemolitoautotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Sulfate reduction(chemolitoautotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Menaquinone
Menaquinone
‐75 mV UbiquinoneMenaquinone +113 mV
Horton et al., Biochemie, 4. akt. Auflage
Redox potentials
ΔG0‘ = ‐ n F ΔE0‘
Redox potential of SO42‐/HSO3
‐ toonega ve → cannot be reduced byNAD(P)H (or ferredoxinred)
SO42‐has to be activated at the
expense of ATPAdenylation of SO4
2‐ to APS
Brock Mikrobiologie © Pearson 2013
Sulfate reduction(chemolitoautotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Qmo = quinone‐interactingmembrane boundoxidoreductase complex
Sulfate reduction(chemolitoautotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Qmo = quinone‐interactingmembrane boundoxidoreductase complex
Dsr = dissimilatory sulfitereductase complex
Sulfate reduction(chemolitoautotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Qmo = quinone‐interactingmembrane boundoxidoreductase complex
Dsr = dissimilatory sulfitereductase complex
Sulfate reduction(chemolitoautotrophic)
Problems:Sulfate transportSulfate activation
Energyconsumingprocesses
1. Transport system2. ATP‐Sulfurylase3. APS‐Reductase (AprBA)4. Sulfite reductase (DsrAB)5. Hydrogenase6. Cytochrome c37. Cytc3: Menaquinone Oxidoreductase/quinone reductase (Qrc Komplex)8. Menaquinol dependent membrane Oxidoreductase (Qmo Komplex)9. Dsr Komplex (coupled to dissimilatory Sulfite reduction)10. Energy conserving Pyrophosphatase11. Pyrophosphatase12. ATP‐Syntetase13. Dithiol/Disulfide protein DsrC
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
Sulfate reduction(chemolitoautotrophic)
André A. Santos et al. Science 2015;350:1541-1545
Sulfate reducers
Incomplete oxidizers
Sulphate reducing reaction ΔG0‘ group
SO42‐ + 4 H2 + H+ → HS‐ + 4 H2O ‐151.9 autotrophs Many sulfate reducers,
e.g. Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)
SO42‐ + Acetate → HS‐ + 2 HCO3
‐
(also: fatty acids, hydrocarbons, aromatic compounds→ Acetyl‐CoA)‐47.6 Complete oxidizers
(heterotrophs)Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)Many others
0.75 SO42‐ + propionate → 0.75 HS‐ + 2 acetate + HCO3
‐ +0.25 H+
0.5 SO42‐ + butyrate→ 0.5 HS‐ + 2 acetate + 0.5 H+
0.5 SO42‐ + lactate→ 0.5 HS‐ + acetate + HCO3
‐
‐37.7
‐27.8
‐80.2
Incompleteoxidizers(heterotrophs)
Desulfovibrio
Three main physiological groups:
Sulfate reductionincomplete oxidation of organic electron donors
APS
HS‐~ 1 ATP
8 [H]
Oxidative part Reductive part
Via Electron transportphosphorylation (ETP)
SO42‐
ATPPPi
AMP
Corresponds to 2 ATP equivalents whichhave to be invested
HSO3‐
2 Lactate
2 Pyruvate
2 Acetyl‐CoA
2 Acetate
2 ATP(SLP)
ΔG0‘ = ‐160 kJ/mol SO42‐ red.
Incomplete oxidation of organic compounds to acetate allow for ATP formation via SLP
2 CO2
APS reductase
Sulfite reductase
Sulfate reduction(chemoorganoheterotrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Qmo = quinone‐interactingmembrane boundoxidoreductase complex
Dsr = dissimilatory sulfitereductase complex
[H] from catabolismFerredoxinred, NAD(P)H,
(MQH2)
Sulfate reducers
Incomplete oxidizers
Sulphate reducing reaction ΔG0‘ group
SO42‐ + 4 H2 + H+ → HS‐ + 4 H2O ‐151.9 autotrophs Many sulfate reducers,
e.g. Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)
SO42‐ + Acetate → HS‐ + 2 HCO3
‐
(also: fatty acids, hydrocarbons, aromatic compounds→ Acetyl‐CoA)‐47.6 Complete oxidizers
(heterotrophs)Desulfobacteriaceae(proteobacteria)Desulfotomaculum(gram positive)Archaeoglobus(archaea)Many others
0.75 SO42‐ + propionate → 0.75 HS‐ + 2 acetate + HCO3
‐ +0.25 H+
0.5 SO42‐ + butyrate→ 0.5 HS‐ + 2 acetate + 0.5 H+
0.5 SO42‐ + lactate→ 0.5 HS‐ + acetate + HCO3
‐
‐37.7
‐27.8
‐80.2
Incompleteoxidizers(heterotrophs)
Desulfovibrio
Three main physiological groups:
Sulfate reductioncomplete oxidation of acetate as organic electron donor
APS
HS‐~ > 2 ATP
8 [H]
Oxidative part Reductive part
SO42‐
ATPPPi
AMP
Corresponds to 2 ATP equivalents whichhave to be invested
HSO3‐
Acetate
Acetyl‐CoA
2 CO2
TCA cycle (modified) orOxidative CODH/ACS pathway
Acetate activationEnergy consuming
Acetate + SO42‐ + 2 H+ 2 CO2 + HS‐ + 2 H2O ΔG0‘ = ‐41 kJ/mol SO4
2‐ red.
Via Electron transportphosphorylation (ETP)
APS reductase
Sulfite reductase
Sulfate reductioncomplete oxidation of acetate as organic electron donor
APS
HS‐~ > 2 ATP
8 [H]
Oxidative part Reductive part
SO42‐
ATPPPi
AMP
Corresponds to 2 ATP equivalents whichhave to be invested
HSO3‐
Acetate
Acetyl‐CoA
2 CO2
TCA cycle (modified) orOxidative CODH/ACS pathway
No ATP via substrate level phosphorylationReduction equivalents: ferredoxin (‐400 mV), NAD(P)H (‐320 mV) andmenaquinone (‐75 mV);APS/SO3
2‐ ‐60 mV, SO32‐/HS‐ ‐110 mV
Acetate activationEnergy consuming
Via Electron transportphosphorylation (ETP)
APS reductase
Sulfite reductase
The Citric acid cycle for complete acetate oxidationin arobic organisms
Two irreversible steps <‐30 kJ/mol
→ Energy dissipation
‐500mV
‐190 mV
+ 30 mV
‐320 mV
+113 mV
‐320 mV
APS/SO32‐ ‐60 mV,
SO32‐/HS‐ ‐110 mV! Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem 176:497‐508
Acetate activation (two ATP equivalents)
Redox potentials
ΔG0‘ = ‐ n F ΔE0‘
Brock Mikrobiologie © Pearson 2013
The Citric acid cyclecomplete oxidation in Desulfobacter postgatei
UQH2
+113 mV ‐450 mV
‐75 mV
‐75 mVMenaquinoneElectron acceptor with morepositive redox potential than NAD+ movesequilibrium towardsOxaloacetate formation
Acetate activation via Succinyl‐CoA: acetate CoA‐transferase (together with ATP‐Citrate Lyase makes the acetateactivation energetically neutral)
‐500mV
‐190 mV
+ 30 mV
ATP‐Citrate lyase (reversible); the exergonic malate‐oxaloacetateconversion allows for energeticcoupling of the Acetyl‐CoA‐oxaloacetate condensation
2‐Oxoglutarate: ferredoxinoxidoreductase(reversible) Use of ferredoxinrenders reaction reversible andallows for optimized energeticcoupling via ETP
Menaquinone (morenegative redox potential thanubiquinone), redox differencebridged via concentration (orconfurcation), enables electrontransfer to APS and SO3
2‐
→ with modifications
Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem176:497‐508
Sulfate reduction(chemoorganoheterootrophic)
Aus Fuchs, Allgemeine Mikrobiologie, 9. Auflage, Thieme
APS reductase Sulfite reductaseATP‐Sulfurylase
Qrc = quinone reductase complex
Qmo = quinone‐interactingmembrane boundoxidoreductase complex
Dsr = dissimilatory sulfitereductase complex
[H] from catabolismFerredoxinred, NAD(P)H,
MQH2
?
Respiratory chaincomplete oxidation in Desulfobacter postgatei
Aerobic organisms Desulfobacter postgatei(proposed)
Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem 176:497‐508
Proposed electron flow in the respiratory chain of Desulfobacter postgatei in comparison to that in mitochondria and aerobes e.g. Paracoccus
→∆µH+ = generation ofelectrochemical protonpotential
The Citric acid cyclecomplete oxidation in Desulfobacter postgatei
UQH2
+113 mV ‐450 mV
‐75 mV
‐75 mV
‐500mV
‐190 mV
+ 30 mV
ATP‐Citrate lyase (reversible); the exergonic malate‐oxaloacetateconversion allows for energeticcoupling of the Acetyl‐CoA‐oxaloacetate condensation
2‐Oxoglutarate: ferredoxinoxidoreductase(reversible) Use of ferredoxinrenders reaction reversible andallows for optimized energeticcoupling via ETP
→ with modifications
Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem176:497‐508
Especially the substitutions ofirreversible Citrate synthase and2‐Oxoglutarate dehydrogenaseby the reversible ATP‐Citrate lyase and Oxoglutarate: ferredoxin oxidoreductase, respectively, make the wholecycle reversible!
→ reversed pathway used forCO2 fixation during autotrophicgrowth
The oxidative CODH/ACS pathwaycomplete oxidation in Desulfotomaculum acetoxidans
‐420 mV
‐117 mV
‐295 mV
‐524 mV
• Acetate activation via acetatekinase and phosphotransacetylase→ 1 ATP instead of two for acetateactivation, regained in CHO‐FH4 toformate conversion
• Menaquinone instead of NAD+
makes the CH3‐FH4 to CH2=FH4conversion reversible
→ pathway is reversible and acts – in the reversed direction – in CO2fixation during autotrophic growth
Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem 176:497‐508
Respiratory chaincomplete oxidation in Desulfotomaculum acetoxidans
Thauer RK (1988) Citric‐acid cycle, 50 years on. Eur J Biochem 176:497‐508
Proposed electron flow in the respiratory chain of Desulfotomaculum acetoxidans
→∆µH+ = generation ofelectrochemical proton potential
X = so far unidentified electron carrier
Sulfur reduction
• Several anaerobes use elemetal sufur S0 as electron acceptor
• Some of them are obligate sulfur S0 reducers (e.g. some hyperthermophilicarchaea like Thermoproteus, Pyrodictium)
• Redox potential S0/HS‐ ‐270 mV
• Others also utilize other electron acceptors e.g. Desulfuromonas acetoxydans, they only induce the sulfur respiratory enzymes on demand
Questions 6
• Describe the three physiological groups of sulfate reducers. Whatare the substrates, what the products?
• What are the (energetic) difficulties in sulfate reduction? Why hassulfate to be activated and how much ATP does it require? What isthe product of sulfate activation? How does sulfate enter the cell?
• How is energy gained in the incomplete oxidation of lactate?• Which pathways lead to complete oxidation of acetate? What is
additionally challenging with respect to energetics during growthon acetate?
• Why does sulfate reducers outcompete methanogens in thepresence of SO4
2‐?• How does sulfate reducers fix CO2 during autotrophic growth with
H2?• Name at least 3 pathways of CO2 fixation!
Questions 6
• Welches sind die drei physiologischen Hauptgruppen der Sulfat Reduzierer? Was sind die Substrate, was die Produkte?
• Was sind die energetischen Hürden bei der Sulfat Reduktion? Warum muss Sulfat aktiviert werden und wieviel ATP wird dazu benötigt?Was ist das Produkt der Sulfat Aktivierung? Wie wird Sulfat in die Zelle aufgenommen?
• Wie wird bei der unvollständigen Oxidation von Laktat Energie gewonnen?
• Über welche Stoffwechselwege wird Acetat vollständig zu CO2 oxidiert? Was stellt beim Wachstum auf Acetat eine zusätzliche energetische Hürde dar?
• Warum sind die Sulfat Reduzierer gegenüber Methanogenen in Gegenwart von SO4
2‐ im Vorteil? • Wie fixieren Sulfat Reduzierer CO2 bei autotrophem Wachstum mit
H2?• Benennen Sie wenigstens 3 CO2‐Fixierungswege.
Übung: Mikrobielles Wachstum II
Wie entwickelt man ein Medium?
Übung: Mikrobielles Wachstum II
1 g TZ/ 1 g C
Minimalmedium (definierte Zusammensetzung), C limitiertes Wachstum (heterotroph)
~50% der C & E Quelle gehen in den Baustoffwechsel
Wie viel Stickstoff (NH4Cl in mM) muss dem Medium mindestens zugegeben werden, damit der Stickstoff bei einer gegebenen C‐ & E‐Quelle (20 mM Glucose) nicht limitierend wird?
Übung: Mikrobielles Wachstum II
20 mM Glucose 0,12 mol C (Glucose 6 C‐Atome) 1,44 g C (C 12 g/mol) 1,44 g TZ
Man benötigt also mindestens 0,175 g N (1,44 x 0,12 (12% TZ sind N) 0,175 / 14 (N 14 g/mol) =12,5 mM NH4Cl ( =0,66 g/l)
1 g TZ/ 1 g C
Minimalmedium (definierte Zusammensetzung), C limitiertes Wachstum (heterotroph)
~50% der C & E Quelle gehen in den Baustoffwechsel
Übung: Mikrobielles Wachstum II
20 mM Glucose 0,12 mol C (Glucose 6 C‐Atome) 1,44 g C (C 12 g/mol) 1,44 g TZ
Man benötigt also mindestens 0,175 g N (1,44 x 0,12 (12% TZ sind N) 0,175 / 14 (N 14 g/mol) =12,5 mM NH4Cl ( =0,66 g/l) ~40 ‐60 mM NH4Cl
Minimalmedium (definierte Zusammensetzung), C limitiertes Wachstum (heterotroph)